Purification and characterization of caffeine synthase from tea leaves

Abstract

Caffeine synthase (CS), the S-adenosylmethionine-dependent N-methyltransferase involved in the last two steps of caffeine biosynthesis, was extracted from young tea (Camellia sinensis) leaves; the CS was purified 520-fold to apparent homogeneity and a final specific activity of 5.7 nkat mg-1 protein by ammonium sulfate fractionation and hydroxyapatite, anion-exchange, adenosine-agarose, and gel-filtration chromatography. The native enzyme was monomeric with an apparent molecular mass of 61 kD as estimated by gel-filtration chromatography and 41 kD as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme displayed a sharp pH optimum of 8.5. The final preparation exhibited 3- and 1-N-methyltransferase activity with a broad substrate specificity, showing high activity toward paraxanthine, 7-methylxanthine, and theobromine and low activity with 3-methylxanthine and 1-methylxanthine. However, the enzyme had no 7-N-methyltransferase activity toward xanthosine and xanthosine 5'-monophosphate. The Km values of CS for paraxanthine, theobromine, 7-methylxanthine, and S-adenosylmethionine were 24, 186, 344, and 21 microM, respectively. The possible role and regulation of CS in purine alkaloid biosynthesis in tea leaves are discussed. The 20-amino acid N-terminal sequence for CS showed little homology with other methyltransferases.

Figure 1

Pathways for the biosynthesis of caffeine. Solid arrows indicate major biosynthesis routes; dotted arrows indicate minor pathways. A XMP → 7-methyl XMP → 7-methylxanthosine pathway is operative in coffee leaves but not in tea leaves. Xanthine is converted to purine alkaloid via a minor route; it is also the entry point in the purine catabolism pathway (based on data reviewed by Ashihara and Crozier [1999]).

Figure 2

Purification of CS from young tea leaves. A, Hydroxyapatite chromatography of proteins precipitated with 50% to 80% saturated (NH₄)₂SO₄. B, Shodex IEC QA-824 anion-exchange chromatography of the active fraction from hydroxyapatite chromatography. C, HiLoad Superdex 200 gel filtration of the elution from adenosine-agarose chromatography. The dotted lines indicate A₂₈₀ and solid circles represent CS activity.

Figure 3

SDS-PAGE analysis of proteins at various stages of purification and photoaffinity labeling of CS from the adenosine-agarose fraction. Fractions from each purification step were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Lane 1, Crude extract (6 μg); lane 2, 50% to 80% saturated (NH₄)₂SO₄ precipitate (23 μg); lane 3, hydroxyapatite (12 μg); lane 4, Shodex anion-exchange chromatography (7.4 μg); lane 5, adenosine-agarose (2.0 μg); and lane 6, Hi-Load adenosine-agarose chromatography (0.7 μg). Lanes 7 and 8, SDS-PAGE after photoaffinity labeling of CS with [methyl-¹⁴C]SAM (1.96 GBq mmol⁻¹). Radioactivity was visualized by an image-analyzer system. Lane 7, With SAH; lane 8, without SAH; lane M, molecular mass marker proteins.

Figure 4

Effect of pH on the activity of tea-leaf CS. CS was assayed in 50 mm Tris-HCl (⋄), 50 mm sodium phosphate (▪), 50 mm Pipes (▿), and 50 mm Mes (•) buffers.

Figure 5

The 20-amino acid N-terminal sequence obtained for CS.